Environ Monit Assess (2007) 131:349364 DOI 10.

1007/s10661-006-9480-6

Bioaccumulation of Metals in Fish of Salmonidae Family and the Impact on Fish Meat Quality
Vildana Alibabi & Nada Vahi & Melisa Bajramovi

Received: 8 April 2006 / Accepted: 22 August 2006 / Published online: 14 December 2006 # Springer Science + Business Media B.V. 2006

Abstract The study was aimed at determining the levels of metals in water samples and muscles of the fish caught in the Una River basin, located in the northwestern part of Bosnia and Herzegovina. For that purpose, three fish species: Brown Trout (Salmo trutta m. fario), Grayling (Thymallus thymallus) and Californian Trout (Salmo gairdneri), together with stem water samples, were analyzed for metal concentrations (Pb, Hg, Cd, As, Mn, Ni, Cu, Cr, Se, Co, Sn, Zn, Fe, Ca, P) during a 2-year period. The fish was captured using electric fishing, nets or fishing equipment. The capture was undertaken on three sites (the river source, the middle flow and the river mouth) of each of the five biggest rivers belonging to the Una River basin (Unac, Krunica, Sana, Klokot, and Una). The concentrations of metals in each sample were determined via atomic absorption spectrophotometry. In the tested waters, the presence of Mn in concentrations higher than permitted

(0.07 mg/l) had been detected. In the tested meat, the following average concentrations of metals (mg/kg) had been found: Pb (0.67), Cd (0.06), Mn (0.65), Ni (0.15), Cu (0.79), Cr (1.05), Se (0.03), Zn (8.92), Fe (5.40), Ca (14.68), and P (10.85). The correlation between Mn concentrations identified in the tested waters and those identified in the meat of Brown Trout was revealed to be statistically significant, which confirms that, over time, bioaccumulation of metals took place. Even though the results were not indicative of contamination, they strongly suggest that constant monitoring of the ecosystems in reference should be implemented. Keywords Bioaccumulation . Environmental pollution . Heavy metals . Salmonidae family . Water quality

1 Introduction
V. Alibabi : M. Bajramovi Biotechnical Faculty, University of Biha, Kulina Bana 2, 77 000 Biha, Bosnia and Herzegovina N. Vahi (*) Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10 000 Zagreb, Croatia e-mail: nvahcic@pbf.hr

Heavy metals are among the most hazardous metals that could be bioaccumulated in a habitat. The fact that they increase in values by passing from lower to higher organisms, is even more disturbing (Hammond, 1971). Research has shown that out of all water dwellers, fish is the one most susceptible to toxic substances present in water. In fish, the process of contamination instigated by water pollutants, takes a slower course than in mammals (Mammalia). Aside

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from providing data on fish health and deviations encountered, and aside from enabling the comprehension of the aforementioned, as well as the apprehension of the effects generated by toxic substances, fish monitoring aids in evaluating the state of water biota. It is, however, of the outmost importance for signalling the pollution of water biota (Zelikoff, 2000). Another two important reasons that justify the need for detecting toxic substances in fish, are, firstly, the fact that fish eventually serves as food and, when consumed, can lead to food poisoning throughout the food chain, and, secondly, atomic spectrophotometric technique shows that the accumulation of toxic substances in water samples is almost regularly near or under the detection limit, so that the research undertaken on a fish model allows for an easier comparison of metal concentrations found in fish habitating different localities (Bols, Brubacher, Ganassin, & Lee, 2001). Expert literature is rich in information on water contamination caused by metals and their effects on fish population. As it seems, heavy metals have been the ones most explored, even in the field of genetics, where cadmium has been proven to be the most hazardous for the fish genetic material. Once Cd contaminates the aquatic ecosystem, it may enter into the aquatic food chain either via direct consumption of water or via biota, including both phytoplankton and zooplankton (Ruangsomboon & Wongrat, 2006). The second most hazardous metal is mercury, while zinc, for example, is the least genotoxic of them all (Snchez-Galn, Linde, & Garcia-Vzquez, 1999). A presence of anionic detergent, for example, causes a decrease in the number of cells that constitute the mucus component and participate in the immune system functioning (Roy, 1988). In case a carp is exposed to cadmium, a decrease in the number of mucus cells is encountered. Exposure of fish to waters that contain Hg, Cd and Cu, leads to the hypersecretion and mucus accumulation. When habitating in waste waters, Salmonidae fish present with a thinner epidermal layer (Burkhardt-Hol, Escher, & Meier, 1997). Bearing this in mind, we can conclude that the contamination of water biosphere takes place all over the world (Almeida, Nocelli, Silva, & Junior, 2001; Alquezar, Markich, & Booth, 2006; Burger, Orlando, Gochfeld, Binczik, & Guillette, 2004; Farkas, Salnki, & Speczir, 2003; Grace, Skaggs, & Cassel, 2006; Jewett et al., 2003; Licata, Di Bella,

Dugo, & Naccari, 2003; Linde et al., 1998, 1999; Luckenbach, Triebskorn, Mler, & Oberman, 2001; Lukin et al., 2003; Maletin et al., 1996; Mansour & Sidky, 2002; Mazet, Keck, & Berny, 2005; Muir, Wang, Bright, Lockhart, & Kck, 2005; Norris, Camp, Maldonado, & Woodling, 2000; Olsvik, Gundersen, Andersen, & Zachariassan, 2001; Reash, Lohner, & Wood, 2006; Turgut, 2003; Usero, Izquierdo, Morillo, & Gracia, 2003; Yamaguchi, Gazzard, Scholey, & Macdonald, 2003; Yilmaz, 2003). When it comes to fish, contaminating effects of water biota are predominantly reflected through a decrease in fish growth and their developmental potential, suppressed or completely thwarted reproduction, characteristic health impairments and large-scale migrations into clean waters, as well as through increased mortality rates, and even the annihilation of entire populations. Heavy metals that reach waters, originate from industrial and waste waters, or from natural or different anthropogenic emissions. The natural environment of the Una River basin comprises areas that pose as manganese and lead zones. Therefore, monitoring and control of the food originating from these areas is very important, especially with regard to the presence of these two metals. On top of the above-mentioned, there exists the problem of usage of different chemical substances in agriculture, the outstanding issue of numerous and unforeseeable manners of waste disposals and unsanitary municipality waste disposal sites, the issue of medical waste, and the issue of a numerous unexploded devices still present on the territory in reference as the residua of the recent war, altogether imposing the need for launching an adequate research (Alibabi, ahinovi, Merdani, Beiraj, & ehaji, 2002). Having in mind that the strategic development of this area relies on the growth of agriculture and food industry, as well as on the advancement of food technology based on domestic resources and the development of river tourism oriented on the Una River and its tributary streams, the significance of ecotoxicological research becomes apparent, and the results to be obtained by such a research prove their manifold use in several aspects of life. Therefore, this research was aimed at determining the condition of the ecosystem of the Una River, with regard to the presence of toxic metals, including that of the top-hazardous ones. The latter refers to Hg, Cd, Pb and the metalloid As. In case of an overdose with metals like Mn, Ni, Cu, Cr, Se, Co, Sn, and Zn,

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perilous effects may occur. Therefore, our research addressed the metals in question, just as it did Fe, Ca and P, which fall into the category of necessary nutrients. Within this frame, possible human exposure to heavy metals and their impact on human health were estimated.

2.2 Sampling procedure Within the frame of this research, three fish species were analyzed: Brown Trout (Salmo trutta morfa fario) belonging to the Salmonidae family, Grayling (Thymallus thymallus) belonging to the Thymalidae family, considered to be the most representative of the rivers under research, and Californian Trout (Salmo gairdneri) as the representative of the imported species, commercially bred in the fish farm. On every sampling point, fish of all three species was sampled, except for the source of the Klokot River, where there exist no living Graylings, allegedly due to the great proportion of Salmonidae family in these waters (the information obtained from the fishery agency Una, Biha). Fish was captured in surface waters at the depths of 02 m using electro-shocking, or was caught in nets. In each river, the fish was captured at three locations (the source, the middle flow, and the river mouth) during the period extending from May/July 2002 to May/July 2003. At the same time, and at the same locations, water was sampled as well. Water samples were randomly collected at the depths of 12 m, stored in glass vials and kept at +4C until the analysis. The fish samples of the three fish species, collected from each river and from each of the three sections of each of the rivers in questions, were weighted in order to determine their average weight (which turned out to be 147.30 g for Brown Trout; 180.21 g for Grayling; and 128.25 g for Californian Trout). The average length of the fish collected in the abovedescribed manner was 23.7 cm for Brown Trout, 22.89 cm for Grayling, and 22.24 cm for Californian Trout. According to the standard and officially adopted methods (AOAC, 2000), in case the fish was longer than 15 cm, a single sample was considered sufficient. If the fish was smaller, it was necessary to engage a minimum of five pieces per analysis (AOAC, Official Method 937. 07). The fish samples were transported to the laboratory in a thermos bag filled with ice on the same day they were collected. When it comes to the fish analyzing, the analysis involved fish muscle samples only. Muscle samples were obtained by working the skin off the fish with a filleting knife. The muscles were then fragmented, homogenized and packed in a polyethylene wrap, and subsequently stored at 20C in order to be preserved for the analysis.

2 Materials and Methods 2.1 Study sites The area subjected to field testing represents the northwestern part of Bosnia and Herzegovina. The surface of the basin equals to 9,368 km2, and is very rich in water. The biggest of its rivers, the Una River, has seven bigger and about ten smaller tributary streams that are known for their flora and fauna diversity, and especially for their richness in fish populations. With its smaller part, the Una River borders upon the Republic of Croatia and partly belongs to our western neighbour. The research included the most important right tributary streams of the Una River: Unac, Krunica, and Sana, as well as the left and most significant tributary, Klokot, which flows into the Una River near the City of Biha. For the purposes of this research, samples were taken from three main sections of each water-flow explored: the source, the middle flow, and the river mouth. The Una River was explored at two localities. The first was situated behind the mouth of the Unac River, which eventually flows into the Una River, i.e., at the 20th km counting from the Una River source. The second was situated behind the mouth of the Krunica River, eventually flowing into the Una River as well, i.e., at the 120th km from the Una River source (approximately 14 areas of water biota). The research included the fish farm built about 100 m downstream from the source of the Klokot River (the Municipality of Biha) (source *IBG Program Office, Swiss Agency for Development and Cooperation). The research included a total of 15 locations. Figure 1 shows all sampling points, as well as bigger settlements by the river basin. According to the information obtained from the Ministry of Social Welfare, the USC government and the Swiss Agency for Development and Cooperation, the Una River basin is dwelled by 350,000 inhabitants.

352 Figure 1 The Una River basin sampling points and bigger settlements by the river basin.

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2.3 Analysis aimed at metal determination All water samples were analyzed directly, without any preparation whatsoever. For the purpose of metal determination, fish muscle samples were digested according to the standard methods (AOAC, 2000). All fish samples were fragmented, homogenized and prepared in the manner compliant with the wet digestion method, capable of detecting all elements of interest, particularly mercury and arsenic. The samples were destroyed using the mixture of nitric/ perchloric/sulphuric acid (3:2:1). The presence of mercury was determined using cold-vapor technique, while the presence of other metals was determined using atomic absorption spectrophotometer, AAS (UNICAM 969), engaging air-acetylene flame and the appropriate hollow cathode tubes. Metal concentrations were calculated using calibration curves (applying the correlation coefficient above 0.99), and the results were expressed in milligrams per kilogram wet weigh. Instrumental detection limit for all metals ranged from 0.003 to

0.01 ppb, except for Hg, As and Se, which can be determined at the levels of 0.1 ppb (Se) and 0.5 ppb (Hg and As). All samples were run in duplicate. The variation coefficient of the replicate analysis ranged from 2%5%. Blanks and standards were run with each sampling batch (fish species). For all samples, blank concentrations were significantly lower than the sample ones. The metal recovery rate ranged from 95 to 105%, and the results have not been corrected in this regard. Standard stock solutions were obtained from Merck. All reagents were of an analytical grade. Phosphorus sample concentrations were determined using UV/VIS spectrophotometer (Model CARY 1.3 VARIAN). The absorption was determined at 440 nm, according to the standard methods (AOAC, Official Method 973.55). 2.4 Data analysis Differences in metal levels, encountered between fish species, rivers and sampling locations engaged on each river, were revealed using a two-way analysis of

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variance (ANOVA), the level of significance thereby being set at 5% (probability limit of P <0.05). In order to relate metal levels encountered in the water samples with metal levels found in the fish muscles, a linear regression analysis had been used. All statistical analyses were conducted using Microsoft Office Excel 2003 software package.

3 Results and Discussion The purpose of this work was to determine the presence of a particular group of metals in the water ecosystem of the Una River basin. Having regard to the possibility of bioaccumulation of these metals in tissues of living organisms, including fish, it was necessary to find out whether the metals determined in water samples were to be accumulated in the fish tissue. Having defined the toxic portion in the fish fillet (edible parts), the risk imposed on a local population was evaluated. The research presented herein had been conducted in the following two major tracks: (a) the determination of metal concentrations in waters; and (b) the determination of metal concentrations in fish fillet samples. In accordance with the ultimate goals, the results are given in two separate chapters. 3.1 Metal concentrations in tested waters Metal concentrations (mean values and standard deviations) established in water samples taken from the rivers listed in the previous text sections, together
Table I Mean concentrations (mg/l) of metals in the tested water Rivers Metal (mg/l) Mn Unaca Klokota Krunicaa Sanaa Unab Xc c Fish farm
a b c

with the results of phosphorus measurements, are presented in Table I We failed to detect any mercury or arsenic as well as Pb, Cd, Ni, Cr, Se, and Co in the waters of the Una River basin. These elements could not be determined either because their actual concentrations are below the instrumental detection level, or because they are not present at all. Elements that are present in the above-mentioned waters are the following: Mn, Cu, Zn, K, Fe, Ca, and Mg (Table I). In two sampling areas, the concentration of manganese exceeds the maximal permissible limits (MRL equals 0.05 mg/l; SFRY, 1987). One of these areas is situated in the middle flow of the Sana River (location 11), where Mn concentrations of 0.07 mg/l were determined. The second one is the area lying at the mouth of the Sana River (location 12), where Mn concentrations of 0.06 mg/l were determined. In other sampling areas, manganese concentrations ranged from 0.001 to 0.05 mg/l. The increase in manganese concentrations, encountered in the Sana River, is the result of the presence of nearby manganese deposits (Sanski Most, Tomorine, and Majkii), i.e., is caused by the geomorphologic soil composition. The concentrations of other elements recovered in the referent samples did not exceed the maximal permissible values. (SFRJ, 1987) The maximal permissible concentration of copper equals to 0.1 mg/l (SFRJ, 1987). In tested waters, its actual concentration ranged from 0.004 to 0.01 mg/l. Currently valid regulations do not provide for the maximal permissible zinc concentration in waters, however, according to the EPA (1995), the latter equals to 0.57 mg/l. Yet, according to different

Cu 0.005 0.009 0.011 0.007 0.006 0.008 0.002 0.009

Zn 0.018 0.022 0.016 0.021 0.020 0.019 0.002 0.023

K 0.531 0.372 0.503 0.572 0.523 0.50 0.076 0.347

Na 2.003 1.325 1.115 4.753 3.057 2.451 1.493 1.162

Mg 32.50 24.18 22.41 22.95 23.42 25.09 4.192 24.22

Fe 0.004 0.007 0.005 0.045 0.018 0.016 0.017 0.01

Ca 133.1 118.3 143.0 106.0 150.8 130.2 18.19 119.2

0.001 0.012 0.011 0.051 0.04 0.023 0.021 0.011

Mean concentrations established on three localities situated on the river in question during a 2-year research period Mean concentrations established on two localities situated on the river in question during a 2-year research period. Mean concentrations and standard deviations (five river sites).

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literature sources, zinc is one of the metals prone to bioaccumulation, and its presence in waste waters and industrial waste causes degrading changes in plants and animals. Greater amounts of zinc tend to accumulate in soils and water biota (especially in fish) in neighbouring urban and industrial areas (Bardonnet & Heland 1994; Mielke, Gonzales, Smith, & Mielke, 1999). As shown on Table I, among waters belonging to the Una River basin, the highest manganese concentrations were found in the Sana River, while the lowest were encountered in the waters of the Unac River. Copper was positive in the water of the Krunica River, but the lowest values were measured in the Unac River. Regarding zinc concentrations measured in the rivers embraced by the Una River basin throughout a 2-year research period, they were mostly similar and did not vary considerably either between each other, or during the study course. In the Krunica River, average concentrations of zinc were lower (0.016 mg/l), while in the Klokot River the highest zinc concentrations were established (0.022 mg/l). Calcium is somewhat represented in the Sana River (0.572 mg/l), but was recovered in the lowest proportion from the Krunica River (0.377 mg/l). In the Sana River, the average sodium concentrations reached the highest levels (4.753 mg/l), while in the Krunica River the lowest values were measured (1.115 mg/l). Iron was found to be represented in approximately equal share in all rivers, except for the Sana River, where its concentrations were ten times higher than in other rivers (0.045 mg/l). In the Unac River, the lowest iron concentrations were established. The highest concentrations of calcium were found in the Una River (150.8 mg/l), and the lowest in the Sana River (106.0 mg/l). Unac is the river in which the highest average concentrations of magnesium (32.50 mg/l) were found, while the Klokot River contains the lowest average magnesium share (22.41 mg/l). Bearing in mind that metal concentrations detected in the rivers of the Una River basin are very low, and none of the tested water biota can be considered contaminated, it is difficult to say which river is in the worst shape. However, if manganese, copper and zinc concentrations in the Sana River are taken into account, we can say that this river is polluted. In comparison to the metal concentrations identified in the Danube River, which is considered

contaminated, we cannot say the same for the rivers belonging to the Una River basin. For instance, in Vojvodina borderline region situated near the Hungarian border, in 1991 manganese concentrations of 7.6 mg/l were identified (Maletin et al., 1996), and in the Danube-Tisa-Danube Channel (DTD) in the same year, these values were 62 mg/l. In the water of the Danube River, other metals were found in the following concentrations: copper (2.8 mg/l), lead (15 mg/l), nickel (1.5 mg/l) and iron (405.2 mg/l). The share of the same metals in the DTD Channel was as follows: Cu (3.0 mg/l), Pb (45 mg/l), nickel (7.5 Mg/l), Fe (719.0 mg/l). Concentrations of cadmium determined in the Channel water are at least of the order of magnitude of 0.5 mg/l. Cadmium concentrations in waters of the Una River basin are hundreds, or even thousands of times lower. In an attempt to find the reasons for zinc and copper presence in the rivers of the Una River basin, contribution of anthropogenic activators of metal mobilization which results in their transfer from sediments into waters, especially during the process of water mixing, must be considered relevant. Zinc reaches waters mostly from domestic waste waters, but also via the release from iron and brass. Industrial waste may contain zinc too. Brass salts are used for protection against pests, but represent also a component of pipes and brazen pots which corrode over time, and through their corrosion, brass is released into natural waters. 3.2 Metal concentration in fish fillet meat Mean values and standard deviations of metal concentrations found in the first and second year of research in the filleted meat of the fish habitating the streams/rivers in question, are shown in Table II. Metal concentrations are expressed for each fish type separately, and their mean values and standard deviations are shown in Table III. In the same table, the results obtained by measurements of metal concentrations in fish fillet of Californian Trout originating from the fish farm, are presented. Statistical evaluation of data on metal concentrations established in the filleted meat of the fish habitating the rivers in question, was carried out using the ANOVA method, and is presented in Table IV. Data are sorted out based on the fish type. Significant differences in the share of particular

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Table II Mean concentrations (mg/kg) of metals found in the fillets of the fish originating from the rivers of the Una River basin Metal (mg/kg) Riversa x s Unac Pb Cd Mn Ni Cu Cr Se Zn Fe Ca P I II I II I II I II I II I II I II I II I II I II I II 0.560.053 0.560.038 0.040.009 0.040.007 0.560.065 0.540.079 0.120.019 0.130.012 0.690.168 0.700.139 0.860.074 0.830.072 0.030.003 0.030.005 9.300.365 9.320.219 4.720.419 4.920.289 13.590.602 13.610.267 10.700.717 10.500.764 Klokot 0.700.164 0.750.151 0.060.005 0.060.006 0.570.041 0.580.036 0.150.023 0.150.016 0.940.045 0.950.062 1.070.076 1.090.061 0.040.005 0.040.005 9.160.449 9.160.438 6.560.429 6.760.212 13.510.352 13.710.269 10.710.898 10.750.559 Krunica 0.650.067 0.650.059 0.070.008 0.070.007 0.720.065 0.700.003 0.150.021 0.160.020 0.770.024 0.790.061 1.210.029 1.170.034 0.030.004 0.030.004 8.680.508 8.690.337 4.940.166 4.880.120 16.280.403 16.110.220 9.850.525 9.860.355 Sana 0.690.068 0.690.052 0.070.013 0.070.008 0.700.070 0.730.048 0.180.020 0.170.007 0.770.041 0.760.039 1.140.077 1.120.071 0.020.005 0.020.005 8.130.076 8.200.089 4.470.239 4.530.178 15.870.322 15.890.226 11.470.738 11.840.326 Una 0.710.079 0.750.041 0.070.009 0.070.005 0.710.144 0.690.109 0.150.034 0.140.016 0.790.131 0.810.085 1.010.166 0.970.080 0.060.016 0.050.014 9.320.638 9.250.525 6.140.637 6.510.526 13.480.655 13.870.216 11.550.442 11.830.296

I The first study year; II the second study year

a

Mean concentrations of metals with the accompanying standard deviations, documented across all study localities and in all three fish species.

metals identified in the meat of Californian Trout in different habitats and different research years, are shown in Table V. Correlations between metal concentrations in water and fillet samples of each type of fish embraced by the research, are shown in Table VI. The dose of a certain metal ingested upon fish consumption, depends on the quantity of fish consumed. Based on a daily consumption of 100 g fish meal, we calculated the permissible concentration, the intake and the risk imposed on humans. Apparently, two of the four most hazardous metals, Hg and As, had not been identified at all (their detectible level equals to 0.0005 mg/kg). The presence of these elements is expected in the areas such as North Carolina, USA, that were heavily exploited during the famous gold rush, mercury thereby being used for gold_extraction (May, Hothem, Alpers, & Law, 1999), or in the Spanish province Asturia, where mercury and arsenic are contained in the soil. In India,

the results of soil research (Chatterjee & Mukherjee, 1999) undertaken near the factory in which brazen acetoarsenite, serving as a pesticide (Paris Green), is produced, reveal a large share of brass (33,900 51,100 mg/kg), arsenic (20,10035,500 mg/kg) and chromium (5,3005,510 mg/kg) in the surrounding soil, which results from the nature of the industrial process. High concentrations of Hg and other heavy metals were documented in headwater streams near the mining centers of La Rinconada and Cecilia, and the Lake Titicaca, Peru (0.060.63 mg/kg) (Gammons et al., 2005), as well as in the sediment encountered in the area of Fundy Bay, Canada, (779 ng/g), all arising from industrial and other human activities (Hung & Chmura, 2006). Concentrations of mercury, methylmercury, arsenic, selenium and cadmium were detected in atmospheric deposits, water and biota of two streams in western Maryland. The results of this study show the importance of water chemistry in determining the bioaccumulation of metals and metal-

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Table III Mean concentrations (mg/kg) of metals found in the fillets of the three fish species dwelling in the Una River basin Metal (mg/kg) Fish speciesa x s Brown trout Californian trout Grayling Californian trout from fish farmb 0.97 0.95 0.06 0.05 0.91 0.90 0.11 0.11 0.96 0.97 1.15 1.13 0.04 0.03 10.3 9.97 6.85 6.77 13.7 13.3 10.35 9.81

Pb Cd Mn Ni Cu Cr Se Zn Fe Ca P

I II I II I II I II I II I II I II I II I II I II I II

0.610.076 0.650.096 0.060.011 0.070.013 0.620.117 0.630.107 0.130.026 0.140.022 0.840.090 0.850.910 1.080.143 1.070.133 0.030.014 0.040.013 8.650.517 8.790.500 5.070.937 5.370.999 14.451.403 14.711.258 11.180.841 11.140.750

0.720.115 0.730.114 0.060.016 0.060.013 0.650.105 0.650.092 0.160.024 0.160.020 0.820.089 0.820.084 1.000.282 1.060.121 0.040.014 0.040.012 9.130.556 9.030.507 5.520.905 5.651.179 14.841.223 14.811.119 10.770.707 10.930.878

0.640.101 0.650.082 0.060.017 0.070.016 0.670.096 0.660.104 0.160.028 0.160.026 0.700.148 0.710.128 1.020.1479 0.980.165 0.030.014 0.030.011 8.870.724 8.860.610 5.240.841 5.541.365 14.671.473 14.621.236 10.451.067 10.611.095

I The first study year; II the second study year

a

Mean concentrations of metals and the accompanying standard deviations, encountered across all study localities and in all three fish species. Mean concentration of metals established in three samples taken from the fish bred in the fish farm (in subsequent study years)

loids into invertebrates and fish (Mason, Laporte, & Andres, 2000). In case of the Una River basin, elements like mercury and arsenic are non-existent, since no industrial activities capable of encouraging their release, or the usage of arsenic-based pesticides, are underway. Yet, bearing in mind the multiple use of arsenic and its compounds, it was necessary to determine the status of the Una River basin. Nevertheless, it is essential to provide continuous ecosystem monitoring, because this region is oriented towards the growth of organic and biological food, which has to be launched into the foreign market. Similar research has been conducted in areas in which the economy is based on tourism, and which are in a great need of preserving the ecosystem, as is the case with the Una River basin. Alike Hg and As, Co and Sn could not be found in the fish meat either. Within the frame of this research,

the share of Pb, Cd, Mn, Ni, Cu, Cr, Se and Zn in the filleted meat of the fish habitating the Una River basin had been conducted, and the presence of each and every of the aforementioned had been confirmed. Lead concentrations determined in the fillet samples of all fish types, ranged from 0.50 to 0.92 mg/kg. These differences in lead concentrations, found in the fish originating from different rivers forming the Una River basin, achieved statistical significance (P 0.05), and are shown in Table IV. The same goes for the differences in lead concentrations encountered between various fish species (P 0.05). Levels of contaminants documented in fish are of a particular interest, because of a potential risk imposed on the humans who consume them. A question arises as to how many fish a man can eat daily without jeopardizing his health due to the intake of lead or other elements. If an adult weighting 70 kg, consumes

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Table IV Significant differences in metal concentrations encountered in various rivers in different study years, and the same for the different fish species in the subsequent study yearsa Metal Significant differences between rivers, observed in different study years Source of variation Lead Cadmium Manganese Nickel Copper Chromium Selenium Zinc Iron Calcium Phosphorus
a b

Significant differences between fish species observed in different study years Source of variation Year of research 3 fish species Year of research 3 fish species Year of research 3 fish species Year of research 3 fish species Year of research 3 fish species Year of research 3 fish species Year of research 3 fish species Year of research 3 fish species Year of research 3 fish species Year of research 3 fish species Year of research 3 fish species Fexp 4.0 34.78b 4.0 1.0 0.0 16.33 1.0 25.0b 4.0 669.0b 0.01 2.16 1.0 3.0 0.02 8.93 18.44 13.85 0.36 2.14 1.96 29.97a

Fexp 2.61 31.48b 0.0 0.0 0.15 48.96b 0.0 13.0b 3.33 224.2b 3.90 116.9b 1.0 35.0b 0.07 382.7b 4.5 144.8b 0.94 156.4b 0.97 46.47b

Year of research 5 rivers Year of research 5 rivers Year of research 5 rivers Year of research 5 rivers Year of research 5 rivers Year of research 5 rivers Year of research 5 rivers Year of research 5 rivers Year of research 5 rivers Year of research 5 rivers Year of research 5 rivers

Significant differences obtained at the significance level of p 0.05 ANOVA, carried out in the bifactorial manner, without replication, statistical analysis of the data presented in Tables II and III.

100 g of fish, which equals to one meal, he/she takes into his/her organism 0.067 mg of lead (the latter representing the average value of lead concentration for all three species analysed within the frame of our study). If we compare data on permitted weekly and daily lead dose that is not perilous to human health, the latter according to Prpi-Maji (1995) being 250 g, one gets to the conclusion that while consuming 100 g of fish caught in the Una River, 26.8% of the maximal permissible daily dose will get into the organism. Within the frame of this estimation, we cannot neglect the fact that other food we consume also contains a certain amount of lead, or the fact that meals are taken several times a day. In the light of the foregoing, this result is much more disturbing, although critical values were not exceeded at any point. The results of this research suggest that lead bioaccumulation into the fish habitating the Una River is constantly taking place, but such an assumption had not been statistically confirmed.

Lead is a highly toxic element, released into the natural environment from different sources. Tetraethyllead is released through exhausted gases. Unfortunately, in the area targeted by this research, mostly older cars without catalysers are being used, which represents one of the possible causes of high lead concentrations in the fish fillet samples analysed. Lead has also been used as a component of explosive devices (BCDNRP, 1998). In the period of 1991 1995, a great amount of various explosive devices had been launched into this area, which contributed to lead concentrations in waters, and subsequently also in fish species dwelling in the Una River basin. Aside from other possible sources, geomorphologic soil composition might be a cause of the increase in lead values, as well. In 1998, the USC area was determined by the Department of Agropedology to have the soil of a quality necessary for growing organic and biological food. This goes for all the localities in the canton, except for Biha-Ilida,

358 Table V Significant differences in metal concentrations found in the fish fillets of Californian trout with regard to different habitats and study yearsa Metal Lead Cadmium Manganese Nickel Copper Chromium Selenium Zinc Iron Calcium Phosphorus
a

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Source of variation Year of research 2 habitats Year of research 2 habitats Year of research 2 habitats Year of research 2 habitats Year of research 2 habitats Year of research 2 habitats Year of research 2 habitats Year of research 2 habitats Year of research 2 habitats Year of research 2 habitats Year of research 2 habitats

Fexp 0.11 245.4b 1.0 1.0 1.0 2,601b 65,535b 65,535b 1.0 841b 0.25 7.56 1.0 1.0 3.50 84.16 0.07 136.11 1.35 51.30 0.29 1.65

ANOVA (bifactorial, no replication), statistical analysis of the data presented in Table III.

b Significant differences, obtained at the significance level of P 0.05.

and two localities near Sanski Most, where manganese and lead concentrations were documented to surpass the permissible ones. Based on this finding, we can conclude that the composition of the natural

environment should be held responsible for the share of lead determined in fish, which is getting higher due to a variety of human activities. Among heavy metals, cadmium belongs to the most hazardous ones, but also to those investigated throughout. Due to its similarities with zinc, these two can be easily replaced in different chemical compounds. The fact that plants absorb this metal via their roots, enabling it to reach the food chain, is very important. The sources of pollution are different in nature, and range from industrial complexes releasing waste waters, to a cigarette smoke. Cadmium concentrations (Table II and III) detected in the filleted meat of the fish originating from the target research areas, ranged from 0.030.09 mg/kg, but the difference in their average proportion with regard to the parental river, fish type and the research year, did not reach statistical significance (P 0.05) (Table IV). However, cadmium concentrations detected in the tissue of the fish originating from the Lake Okeechobee, south central Florida, were ten times lower than those observed within this study (Burger et al., 2004). According to the 1988 JECFA recommendation (WHO, 1993), cadmium intake of 0.42 mg/60 kg a week is not considered perilous for adults. If we recalculate the maximal cadmium concentrations determined in the samples of the trout originating from the Sana River (0.09 mg/kg), we can conclude that weekly consumption of 100 g fish caught in the Una River basin leads to cadmium intake of 0.063 mg, which represents 15% of the permitted dose. It is obvious that these values are below the permitted ones, but when it comes to this metal one

Table VI Correlation coefficients established between the concentrations of metals found in the water and in the fish fillets, across all rivers of the Una River basina Abscissa Ordinate Correlation coefficients BT(n= 14) I Mn in the water Cu in the water Zn in the water Fe in the water Ca in the water Mn in the fish fillet Cu in the fish fillet Zn in the fish fillet Fe in the fish fillet Ca in the fish fillet 0.52b 0.16 0.35 0.13 0.30 II 0.66b 0.24 0.46 0.24 0.30 CT(n=14) I 0.05 0.34 0.31 0.13 0.30 II 0.13 0.36 0.22 0.28 0.25 G(n=fs13) I 0.18 0.22 0.41 0.09 0.02 II 0.18 0.23 0.40 0.23 0.03

I The first study year; II the second study year BT brown trout; CT Californian trout; G grayling; n number of samples
a b

Statistical analysis of the data presented in Tables I and II. Significant differences, with the level of significance set at p 0.05.

Environ Monit Assess (2007) 131:349364

359

should be careful. If we compare our results with the levels of cadmium recovered in the muscles of cultured sea bass (Dicentrarchus labrax) originating from the Tyrrhenian Sea (Messina, Italy), which ranged from 0.090.13 mg/kg, and knowing that this fish represents a half of the national eurialine fish farming production (Dugo, La Pera, Bruzzese, Pellican, & Turco, 2006), there is no danger in fish consumption. Manganese was detected in the fish fillet as well (Table II). The highest concentration of manganese was detected in the second year of research, in the fillet of the fish originating from the Sana River (0.730.048 mg/kg), which was to be expected having in mind that the concentrations of this metal were actually highest in the water samples taken from the Sana River, so that the possibility of its bioaccumulation in the living organisms should be allowed by all means. The concentration range determined in the target research areas, including the Sana River, expanded from 0.42 to 0.87 mg/kg meat. Higher share of manganese was determined in the Una water samples in the second year of research, as well. Besides already-mentioned manganese and lead deposits situated near Sanski Most, there are two additional manganese deposits on 110th km of the Una River, so that the increased manganese values determined in these waters were to be expected. Differences in manganese concentrations found in the filleted meat of the fish originating from different rivers, reached statistical significance (P 0.5). Average manganese concentrations determined in the meat of all of the fish subjected to analysis, was 0.65 mg/kg. Manganese concentrations detected in the sediment samples taken from the Dniester River, Moldova, were significantly higher (170 g/g), and the authors compared them to the concentrations detected in the sediments of the Po River, Italy (101187 g/g). In both cases, the presence of manganese was the result of industrial activities, so that high manganese concentrations were to be expected (Sapozhnikova, Zubcov, Zubcov, & Schlenk 2005). The Una River basin is situated in the manganese-rich area, so that high concentrations of this metal are to be expected as well. However, with the consumption of 100 g of the fish meat containing the highest manganese share determined within the frame of this research (0.89 mg in the samples of Californian Trout coming from the Una River,

location 14 situated nearby the above-described manganese deposit), the intake of manganese equals to 2.5% of the permitted daily dose. It is obvious that the consumption of this fish does not lead to the excess in permissible daily dose, and that manganese in the present concentrations does not pose as a risk for the state of the habitat and the people living there. Nickel was determined in the filleted meat of the tested fish as well, ranging in concentrations from 0.110.21 mg/kg (Table II). Statistical evaluation revealed nickel concentrations in the fish meat samples to be significantly different from each other (P 0.05), based primarily on the parental river, but also on the particular fish type. Data on permissible nickel shares in the fish meat are indigent, but U.S. EPA (1980) proposed the daily dose of 1.46 mg/kg. It also adduces that the concentration above 50 mg/kg is toxic for plants. The average value encountered in the filleted meat of the fish originating from the rivers of the Una River basin, was 0.16 mg/kg. That means that the consumption of 100 g of fish meat results in the intake of 1.03% of the permitted dose, which is far below the critical level. Copper is a biogenic element necessary for supporting the metabolic processes of the living organism. However, its excess beyond permissible can induce containment in the liver, kidneys and cornea, which results in anaemia. NRC (National Academy of Science, 1980) proposed the permissible daily dose of 23 mg for adults, or, in other words, 0.05 mg/kg body mass, which in case of a 70-kgweighting man equals to 3.5 mg. Copper concentration found in the fish originating from the Una River basin averaged 0.79 mg/kg. Provided that a person consumes 100 g of the fish meat coming from the Una River per day, his/her daily cooper intake would not exceed the permitted values, because the concentration presumed to be ingested in this manner equals to 2.3%. In some aquatic organisms dwelling in the Pearl River Estuary, China, or in some Norwegian rivers, the concentrations of copper were found to be double that (1.81 and 1.30 mg/kg, respectively) (Ip, Li, Zhang, Wong, & Zhang, 2005), which was also the case in some fish caught in Saricay, South-West Anatolia, Turkey (0.074.36 g/g) (Yilmaz, zdemir, Demirak, & Tuna, 2007). In addition, higher concentrations of copper (0.9492.1 g/g) were registered in Portugal, in the Tagus Estuary situated on the European Atlantic coast (Frana, Vinagre, Cacador,

360

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& Cabral, 2005), and in ten sites located along the Drme River (Rhne-Alpes region), the latter ranging from 3.16 to 13.99 mg/kg (Mazet et al., 2005). When it comes to chromium, human potential of absorbing this metal is very weak, so that only 3% of the exposure dose ends up in the human body. The greatest chromium share is found in the spleen, kidneys and testicles. Even the exposure to low chromium concentrations can lead to skin irritations and tissue lesions, while higher concentrations cause kidney and liver damage and damages to the cardiovascular and nervous system (Dunne, 1996). Chromium concentrations found in plants, and relating concentrations found in meat and other animal food products, depend on the composition of soil out of which chromium may reach a water system. Estimated daily intake ranges from 80 to 100 g. According to the Institute of Medicine (2001), in case of an adult the RDA for chromium equals to 200 g. According to the results obtained by this study, chromium concentrations in the meat of the fish originating from the Una River basin, range from 0.68 to 1.29 mg/kg. In case of a 100-g daily consumption, the intake of chromium equal to 0.104 mg can be expected, which represents 52% of the permitted daily dose. Selenium concentrations in the filleted meat of the fish coming from the Una River basin, ranged from 0.010.07 mg/kg. According to the RDA (WHO, 1980), the permitted human daily dose of selenium equals to 0.0015 mg/kg, which means that an adult weighting 70 kg may take 0.01 mg of selenium per day. The fish meat sampled at study locations contained 0.03 mg of selenium/kg (0.003 mg/kg/ 100 g of meat), which means that we ingest 3% of the permitted selenium dose in one course. Selenium is largely present as an industrial polluter. It is released as a side product of metal, glass, plastic and color production in rubber industry. Even though the target research areas lack such industries, the industrial products released by these industries are in use and contain selenium. Bearing in mind that the territories of both Bosnia and Herzegovina and Croatia embrace soil poor in selenium (Beker, 1993), its presence might be attributed to the impact of different waste deposits and their elutriation. Zinc is an essential element present in the human body in a considerable amount, equal to 1.8 g. Marked concentrations of Zn were detected in the tissues sampled from the fish dwelling in the Dipsiz stream

of the Yagatan basin, southwestern Turkey. Mean Zn concentrations ranged from 1.06 to 55.41 mg/kg (Demirak, Yilmaz, Tuna, & zdemir, 2006). In the southwestern Ohio, the concentrations of Zn in the tissue taken from the fish habitating the Campaign Creek stream, ranged from 75.31 to 442.54 g/g, and are considered very high (Reash et al., 2006). According to the NRC, daily dose permitted to be taken by a male equals to 15 mg, and the one to be taken by a female to 12 mg. The average concentrations recovered in the caught fish equaled to 8.89 mg/kg. With the consumption of 100 g of the fish meat containing zinc concentration of this order, an adult ingests 5.93% of the daily permissible dose. Iron, calcium and phosphorus, all falling into the category of the essential elements, were searched for in the fish fillets as well (Tables II and III). The differences in their concentrations, encountered in the filleted meat of the fish stemming from different rivers of the Una River basin, reached statistical significance (p 0.05), while in various fish species only the differences in phosphorus concentrations managed to achieve the significance of that kind. Consumption of 100 g of fish meat provides for 0.18% of the recommended daily dose (National Academy of Science, 1980) of calcium, 5.4% of the iron dose recommended for men and 3.8% of that recommended for women, and only 1.33% of the daily phosphorus requirement, which confirms that the fish originating from the Una River can not provide for the satisfactory amounts of calcium, iron and phosphorus, capable of meeting the needs of the population living in the tested area. While comparing the results obtained regarding metal concentrations in the meat of Californian Trout originating from the natural habitat, and the one being bred in the fish farm (Table III), the significant difference (p 0.05) in lead, manganese, nickel and copper concentrations had been revealed, as well as the significant difference in nickel concentrations observed in different study years (Table V). Burger and Gochfeld (2005) tested 11 types of fish obtained from the markets of New Jersey and analyzed them for arsenic (0.233.3 ppm), cadmium (0.00013 0.03 ppm), chromium (0.040.34 ppm), lead (0.04 0.34 ppm), manganese (0.150.98 ppm), mercury (0.010.65 ppm), and selenium (0.051.02). The authors suggest that more information on the levels of contaminants found in fish should be provided, so

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that people could make a decision what fish to eat to reduce the risk from contamination. Fisher s coefficients, obtained by the statistical evaluation of the study data, demonstrated beyond any doubt that in the different rivers of the Una River basin, and in different fish species, metals are present in different ratios (Tables IV and V). Regarding the year of research in which the data were obtained, no statistically significant difference in the fluctuation of metal concentrations had been revealed (p 0.05). In their research, Linde et al. (1998) obtained similar results, which leads to the conclusion that a trout serves as a good fish model for the biomonitoring of heavy metals, especially copper and lead. Metal concentrations in the fish originating from the Una River basin are mostly lower than those provided in the referral literature. In comparison, in their work Linde et al. (1998) determined copper concentration of 13.57 mg/kg, zinc concentration of 29.01 mg/kg and lead concentration of 0.17 mg/kg. Comparing these metal concentrations with those found in the fish dwelling in the Una River basin, we can conclude that copper and zinc concentrations found in the fish meat stemming from the Spanish rivers Esva, Nora and Piles, are ten times higher than in those stemming from the Una River basin, but, at the same time, in the latter lead concentrations are five times higher. From an ecotoxicologic point of view, this comparison puts in doubt the general opinion that waters of the Una River basin are clean and unpolluted. Similar research, conducted by the Agricultural Department (SAD) on several rivers located in the area of New Mexico, showed metal concentrations in the trout to be as follows: copper, 3.38 g/g; arsenic, 0.03 g/g; cadmium, 0.36 g/g; zinc, 20.88 g/g; mercury, 0.04 g/g; selenium, 6.62 g/g; and lead, 0.27 g/g. A total of 17 elements were analysed. The highest lead concentration was detected in the kidneys and liver of the trout habitating the Terrero area (1.48 g/g), which leads to the conclusion that, under such circumstances, fish has been exposed to chronic lead poisoning. In conclusion, the authors indicate that As, Cd, Cu, Zn, Hg and Se concentrations encountered in the area, do not pose as a risk to the population living there. They adduce, however, that lead concentration in the tested mammals equals to 3.79 g/g tissue, while the average concentration in the fish tissue is 0.27 g/g , meaning that they both

surpass the referent values proposed within the NCBP (National Containment Biomonitoring Program), and may represent a danger to the plants and animals populating this area, including people, as well. The estimation of metal concentrations in sediments provides better knowledge on metal bioaccumulation (Gai, 1996; Linde et al., 1998; Usero et al., 2003; Yilmaz, Erbatur, & Erbatur, 2003), and enables the establishment of a more reliable correlation between metal concentrations found in water, sediment and fish (i.e., their particular organs). Within the frame of this study, an attempt to establish such a correlation was not made. Anyhow, the issue in question represents an additional problem in detecting and elaborating metal bioaccumulation. Since all of the studies concerned with polluted areas provide correlations between metal concentrations established in water and those established in tissues of living organisms, and tend to determine the strength of such a correlation based on the correlation coefficients obtained, the same had been done in this work. Metal concentrations found in the tested waters and in the tested fish meat were put in correlation. Due to the fact that only a few metals could be detected in the tested waters (Mn, Cu, Zn, Fe, Ca), the possibility of determining the correlation coefficient was limited to the aforementioned. The calculated correlation coefficients referring to the metal concentrations found in the particular fish species in each study year, are shown in Table VI. Correlation coefficients related to the concentrations of Ca, Cu, Zn and Fe in the waters of the Una River basin and those in the fish caught at the same locations, did not reach statistical significance for any of the fish species. However, in case of manganese, the determined correlation proved statistical significance, so that metal accumulation in the fish can be proclaimed. In the 1st study year, the calculated correlation coefficient related to manganese concentration equaled to r=0.52, while in the study second year the value of the coefficient in reference was r= 0.66. Both calculated coefficients refer to Brown Trout (S. trutta m. fario). Regarding other two tested species, i.e., Californian Trout and Grayling, no significant correlation had been determined. On the basis of metal concentrations obtained in fish during the course of such a research, precautions to be taken in order to prevent the contamination of the water habitat can be laid down. The top priority is

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the prevention of chromium contamination, followed by that of lead, and finally cadmium. With a regular daily consumption of 100 g of fish meat, 52% of the permitted dose of chromium, 26.8% of the permitted dose of lead, and 15% of the permitted dose of cadmium, are ingested, which certainly stresses the importance of a regular control of the fish and periodical control of the water habitat. With a regular water habitat monitoring, it is possible to timely spread alarming reports on the habitats state, and try to stop additional contamination with these two metals. Other elements analysed within this study did not prove positive in alerting concentrations, although the fact that they are indeed present is a reason enough to provide a constant control of both the fish and the habitat populated by humans.

identification of possible contamination and the prevention of its adverse effects. Metal concentrations established in the water and in the fish meat, were put in correlation. When it comes to Ca, Cu, Zn and Fe, no such correlation was established in any of the fish species targeted by the study. The correlation coefficient calculated for manganese reached statistical significance in both study years, but only in case of Brown Trout (S. trutta m. fario). Nevertheless, such a finding confirms the thesis on metal bioaccumulation in the fish habitating the rivers of the Una River basin.

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4 Conclusions Whereas the presence of Pb, Hg, Cd, As, Ni, Cr, Se and Co failed to be documented, and the concentrations of Zn, Cu, Na, Ca, Fe, K, Mg were revealed to be low and within the permitted boundaries, the waters of the Una River basin can not be considered contaminated. The concentrations of manganese proven in the Sana River at two sampling locations, were above the maximal permissible levels, which is attributed to the geomorphologic source. The research on metal concentrations present in the meat of the fish dwelling in the Una River basin, showed that out of four most hazardous elements, Hg and As were not identified at all. Besides Hg and As, also no Co and Sn, which are very toxic and easily obtained, were detected. The presence of the following elements was determined: Pb, Cd, Mn, Ni, Cu, Cr, Se, Zn, Ca, Fe, and P; but the concentrations encountered were unexceptionally below the permitted ones. Based on the results obtained regarding metal concentrations in the fish, which serve as a basis for the proclamation of the state of alert and calls for precautions to be taken in order to prevent the contamination of the water habitat of the Una River basin, the major role is played by chromium, followed by lead, and finally cadmium. Other elements embraced by this research did not achieve such concentrations, although their presence is a reason enough to provide a constant control of both the fish and the water habitat, which allows for timely

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